1. Field of the Invention
The present invention relates to centrifuge rotors and, more particularly, to centrifuge rotors which support centrifuge tubes at an angle to the spin axis for density gradient separation.
2. Description of Related Art
Essentially, a centrifuge is a device for separating particles suspended in a solution. A centrifuge includes a rotor which supports several containers of sample solution for rotation about a common spin axis. As the rotor spins in the centrifuge, centrifugal force is applied to each particle in the sample solution; each particle will sediment at a rate which is proportional to the centrifugal force experienced by the particle. Centrifugal force is dependent on the mass of the particle, the rotational speed of the rotor, and the distance of the particle from the spin axis. The viscosity and density of the sample solution also affects the sedimentation rate of each individual particle. At a given centrifugal force, density and liquid viscosity, the sedimentation rate of the particle is proportional to its molecular weight, and the difference between its density and the density of the solution.
One of many methods of centrifugal separation is by isopycnic separation, a form of density gradient centrifugation. Such a method permits the separation of several or all of the particles in a sample mixture according to their densities. The method involves a supporting column of fluid (hereinafter referred to as "density gradient fluid") whose density encompasses the whole range of densities of the sample particles and increases toward the bottom of the centrifuge tube. The density gradient fluid typically consists of one or more suitable low molecular weight solute in a solvent in which the sample particles can be suspended. Upon centrifugation, each particle will sediment only to the position in the centrifuge tube at which the density of the density gradient fluid is equal to its own density, and there it will remain. The isopycnic technique, therefore, separates particles into zones or bands solely on the basis of their density differences, independent of time.
Density gradients have been used extensively in the separation and purification of a wide variety of biological materials. For example, nucleic acids have been studied extensively by density gradient methods. For purposes of discussion, isopycnic banding type density gradient centrifugation techniques will be discussed below in connection with DNA banding. In the past, cesium chloride has been successfully used as the density gradient fluid in DNA banding. Under the influence of centrifugal force, the cesium chloride salt redistributes in the centrifuge tube so as to form the required concentrations to create a density gradient. This is often referred to as the self-generating gradient technique in which a continuous density gradient is obtained at equilibrium when the diffusion of cesium chloride towards the spin axis balances the sedimentation away from the spin axis at each radial location along the centrifuge tube.
A nucleic acid may be separated into plasmid DNA and chromosomal DNA by using the cesium chloride density gradient. In addition RNA and proteins in the nucleic acid are separated. The plasmid DNA is separated from the chromosomal DNA by their differences in buoyant density, the plasmid DNA being more dense. More particularly, the plasmid DNA and chromosomal DNA are isolated into isopycnic bands at different radial positions from the spin axis, the plasmid DNA being more dense forms a band at a larger radial distance from the spin axis. In addition, RNA which is heavier forms a pellet at the furthermost radial location in the centrifuge tube and proteins being the lightest particles are "floated" to the innermost radial position close to the spin axis to form a pellet. The RNA and protein are usually not of interest to DNA studies and undesirable as they are a source of contamination of the DNA bands.
In most laboratories, density gradient centrifugation of nucleic acids is carried out using conventional swinging-bucket, fixed-angle and vertical tube rotors. In a swinging bucket rotor, centrifuge tubes are hingedly supported. As the rotor rotates, the centrifuge tubes swing radially outward from a vertical position to a horizontal position. After a period of time, as shown in FIG. 1A, the nucleic acid contained in the centrifuge tubes 18 separates into the plasmid DNA 10 and chromosomal DNA 12 bands as well as RNA 14 and protein 16 pellets. Since the density gradient is formed radially outward from the spin axis, the bands are parallel to the spin axis 20. After centrifugation, the centrifuge tubes 18 return to their vertical position as shown in FIG. 1B. The fractionated DNA bands are extracted from each centrifuge tube using suitable tools. It has been found that nucleic acid separation carried out using a swinging bucket rotor requires long run time to allow sedimentation to take place along the length of the centrifuge tube as indicated by arrow 19. Furthermore, it requires high rotor speeds in order to provide enough centrifugal forces to effect separation of the components located close to the spin axis 20. For a given maximum radial tube position from the spin axis r.sub.max, the average radial distance from the spin axis r.sub.average is substantially shorter thus giving rise to a smaller overall centrifugal force at a given rotor speed.
In a vertical tube rotor, sealed centrifuge tubes have been used in the past such as the Quick Seal.RTM. tubes developed by Beckman Instruments, Inc. as shown in FIG. 2A are supported vertically during centrifugation. Upon centrifugation, the isopycnic plasmid 22 and chromosomal 24 bands and protein 26 and RNA 28 pellets are oriented vertically or parallel to the spin axis 30. After centrifugation, the DNA bands 22 and 24 reorientate into horizontal layers as shown in FIG. 2B. The RNA and protein pellets 26 and 28, however, tend to remain stuck to the centrifuge tube wall. It will be appreciated that the transition of the DNA bands during reorientation from the vertical position shown in FIG. 2A to the horizontal position shown in FIG. 2B causes intermixing of the DNA bands and the pellets as the DNA bands 22 and 24 sweep across the protein and RNA pellets 26 and 28, thereby resulting in contamination of the DNA bands. Furthermore, the protein and DNA pellets may detach from the tube walls when the rotor is at rest and mix the contents in the tube. Precentrifugation clean-up steps such as differential centrifugation will be necessary to remove the protein and RNA particles prior to density gradient separation of DNA bands in order to avoid such contamination. The additional clean-up steps are time consuming. The advantage of vertical tube rotor over swinging bucket rotor, however, is in the increased effectiveness for density gradient centrifugation which in many instances yielding separations in considerably less time than achieved in swinging bucket rotors operating either at the same speed or higher speeds. The centrifuge tubes being vertical in a vertical tube rotor are disposed at a larger average radial distance r.sub.average from the spin axis when compared to a swinging bucket rotor having the same maximum radial tube position r.sub.max. Also, the particle sedimentation path length radially outward across the width of the centrifuge tube as indicated by arrow 31 is considerably less than that along the length of the centrifuge tube in the swinging bucket rotor as shown in FIG. 1B.
The fixed angle rotor is effectively a compromise between the swinging bucket rotor and the vertical tube rotor. The centrifuge tubes 32 in a fixed angle rotor are supported at a fixed angle in the range of 20.degree.-40.degree. to the spin axis during centrifugation, as illustrated in FIG. 3A. Isopycnic DNA bands 34 and 36 and pellets 38 and 40 are formed parallel to the spin axis upon centrifugation. Upon termination of centrifugation and removal of the tubes 32 from the rotor, the DNA bands 34 and 36 reorientate to a horizontal position as shown in FIG. 3B. The probability of contamination of the isopycnic bands 34 and 36 during reorientation is reduced in the case of the fixed angle rotor. However, for a given rotor speed and maximum radius r.sub.max, fixed angle rotors are inherently less efficient than vertical tube rotors due to shorter average centrifuge tube radial distance r.sub.average from the spin axis 42 and increased sedimentation path length as indicated by arrow 43 for a given tube size.